CUADRO 1-16 INDICADORES DE PROCESOS PARA LA ESTRATEGIA DE INTIMIDAD CON LA CLIENTELA

A nim als were sacrificed through a two stage procedure which involved asphyxiation under an increasing concentration of carbon dioxide, followed by cervical dislocation. The abdominal skin was removed and FHT (25 ml per rat) supplemented with heparin (5 units/ml) was injected into the peritoneal cavity, taking special caution to avoid penetration of the gastro-intestinal tract. The abdomen was gently massaged (3 min), before a mid-line incision was completed. Peritoneal lavage fluid was recovered using a plastic pipette and deposited into a disposable polystyrene test tube. Any sample contaminated with blood was discarded. The cells were pelleted by centrifugation [100 g; 2 min; room temperature (RT)], washed twice in FHT and then either purified or used in functional studies directly.

2.4.1.1 Purification of RPMC

Mixed RPMC (as above), to be purified through Percoll were resuspended in BSA-FHT (1 ml). A Percoll solution (4 ml) consisting of 9 parts Percoll and 1 part 10 X CMF was mixed with the cells. BSA-FHT (1 ml) was then layered over

the Percoll/cell mixture, so that a clear interface was produced. The sample was centrifuged (140 g; 25 min; 4 °C) and the supernatant aspirated to leave a pure mast cell pellet at the bottom of the tube. The pellet was then washed twice in BSA-FHT and twice in FHT before use. The yields and purities of RPMC (expressed as percentage valu es-se e section 2.5) w ere also calculated.

2.4.2 Guinea pig iung mast ceils

The anim al was sacrificed w ithout the use of anaesthesia by cervical dislocation. The abdomen was shaved and the skin dissected free. The animal was opened along the midline and the lungs removed from the chest cavity. After dissection to remove the major airways, lung tissue was rinsed in FHT and then cut into small fragm ents (1-2 mm) with scissors and an automated (Mcllwain) tissue chopper. This was followed by digestion (37 ° C; 60 min; with stirring) in FHT supplem ented with BSA (1 m g/m l) and collagenase (120 units/ml). Once the incubation was complete, the digested tissue was further disrupted by expression through a 20 ml syringe. The cells were then filtered through cotton gauze and recovered by centrifugation (150 g; 3 min; 4 °C ). Finally, the cells were washed three times in FHT and then employed in functional studies.

2.4.3. Human iung parenchymal mast cells

M acroscopically normal human lung parenchyma was recovered following surgery for bronchial carcinoma. The specimen was dissected free of pleural tissues, m ajor airways and blood vessels. The sam ple was then crudely chopped into fragm ents (ca 1 cm 3) with scissors and thoroughly washed with FHT. The tissue was chopped once more with a second portion of FHT

(ca 1 litre). The lung tissue was digested in BSA-FHT containing collagenase (160 units/ml) and magnetically stirred in a tissue incubator (90 min; 37 °C). After this digestion, the remaining lung tissue was disrupted by expression (ca 15-20 times) through a syringe (50 ml) and the suspension filtered

through cotton gauze. The filtrate was centrifuged (150 g; 10 min; 4 °C), the cell pellets resuspended in FHT, the supernatants returned to the remaining undigested lung fragm ents and the above digestion process repeated. On confirm ation of the fact that no mucus-like debris remained, the cells were centrifuged (150 g; 3 min; 4 °C) and resuspended in BSA-FHT before finally washing and resuspending for use in functional studies.

2.4.3.1 Purification of human parenchymal mast cells

Isolated human lung mast cells derived from the technique highlighted above could be purified using counter-current centrifugal élutriation (CCE) (348) and percoll gradient centrifugation (349) methods. In this manner, dispersed lung mast cells could be enriched to > 65 % purity with CCE alone. The success of this procedure was dependent on (a) the con centratio n and type of contaminating cells and (b) the amount of m ucus-like factors present which could hinder purification due to cell adhesion.

A suspension of isolated cells were pumped into a Beckman counter-current é lu triatio n cham ber (1000 rpm; 12 m l/m in). The buffer flow rate was increased (25 ml/min), inducing cells of varying size and density to be collected (100 ml) at different intervals. Red blood cells and small, nucleated cells were eliminated at lower flow rates, whereas the bulk of mast cells were collected at higher ones or m erely remained in the élutriation cham ber. Samples at all flow rates were gathered, including those left in the élutriation chamber. These cells were centrifuged and resuspended in FHT (2 ml) for staining (alcian blue) purposes. Overall, flow rates > 20 ml/min rendered sufficiently enriched mast cells to guarantee that these fractions could be pooled.

Elutriated mast cells that were < 60 % pure were enriched further by Percoll gradient sedimentation. In this protocol, Percoll dilutions of 80, 60, 50 and 40 % were composed from the 90 % stock using CMF buffer. Originating with 90 % Percoll, layers of decreasing Percoll density were added (5 ml) to a plastic vial. The sem i-enriched m ast cells w ere ce n trifu g e d and the p e lle t resuspended in the lowest (40 %) Percoll concentration, prior to layering over the 60 % Percoll fraction. This tube was centrifuged (200 g; 20 min; 4 °C ), whereby the cells that had sedimented to the various Percoll interfaces were removed and washed several times. Each cell fraction was counted and

purified mast cells (> 65-95 %) were often found at the 50-60 % and 60-80 % Percoll interfaces. The cells were then diluted in the appropriate volum e of FHT and employed without further purification.

2.4.4 Human BAL mast cells

The BAL technique employed varied slightly depending upon the venue of bronchoscopy, but followed a basic protocol. In brief, after sedation, routine b ro nch oscop y was perform ed using an O lym pus BF 1TR fib re o p tic bronchoscope using 2 % lignocaine fo r local a n a esthesia. BAL was perform ed in the lung opposite to any localised bronch o sco p ica l or radiological abnorm ality using the medial segments of the right middle or lower lobe. After wedging the bronchoscope in the segmental orifice, 3 x 40 ml pre-w arm ed norm al saline was instilled and the cell suspension recovered by suction into a siliconised glass bottle. The fluid was centrifuged at 20 °C (200 g: 4 min) to avoid exposing recovered cells to sudden tem perature fluctuation. The cells were then w ashed tw ice in FHT and employed in functional studies.

2.4.5 Human basophil leucocytes

Blood was collected from healthy volu ntee rs by a q u a lifie d m edical practitioner. As soon as the required volume of blood (20 ml) had been extracted, heparin (50 units/ml) was added to each sample to guard against coagulation. One part (5 ml) of dextran (6 %) containing glucose (30 mg/ml) was mixed with 4 parts of blood and the mixture left to stand for 60-90 min at RT. The top layer (which was rich in platelets and leucocytes) was gently recovered by aspiration and centrifuged (150 g; 3 min; RT) in order to sediment the basophils, leaving the platelets suspended in the supernatant. The supernatant was discarded, the cell pellet washed tw ice in FHT and thence employed for functional studies.